1. Field of the Invention
This invention relates to a member of the guanylate-binding protein family designated as GBP-4 and the cloning and expression of nucleic acid encoding this protein. The invention further relates to methods of production of the isolated molecule and its uses.
2. Description of Related Disclosures
One approach to understanding the molecular basis of cancer is to identify differences in gene expression between cancer cells and normal cells. Strategies based on assumptions that steady-state mRNA levels will differ between normal and malignant cells have been used to clone differentially expressed genes. Zhang et al., Science, 276: 1268–1272 (1997). The recent development and successful application of such strategies include the use of representational difference analysis (Braun et al., Mol Cell Biol 15: 4623–4630 (1995); Lewis et al., Mol Cell Biol 17: 4967–4978 (1997)), serial analysis of gene expression (Velculescu et al., Science, 270: 484–487 (1995)), and quantitative hybridization analysis of arrayed cDNA's. Schena et al., Science, 270: 467–470 (1995).
Cancer of the stomach is a leading cause of cancer deaths worldwide. Parsonnet, Gastroenterol Clin North Am, 22: 89–104 (1993); Peddanna et al., Anticancer Res, 15: 2055–64 (1995). The development of intestinal-type gastric cancer is characterized by successive histopathologic changes progressing from normal mucosa to gastritis to metaplasia and eventually to dysplasia. Stemmermann et al., Hum Pathol, 25: 968–981 (1994).
Cells treated with interferons respond to this stimulus by producing a set of proteins believed to serve as intracellular mediators of the various effects of these cytokines. Interferon-induced GBP's are members of the G protein superfamily and form a distinct subgroup based on their large size (65–67 kDa), potent induction of interferon-γ, and relaxed nucleotide binding. specificity. Nantais et al., J Leukoc Biol, 60: 423–431 (1996). Two other subgroups of GTP binding proteins which are induced by interferon include the Mx proteins, which are involved in the antiviral response (Horisberger et al., J Virol, 64:1171–81 (1990)), and the inducibly expressed GTPases (IGTP's) which are endoplasmic reticulum GTPases that may be involved in protein processing or trafficking. Taylor et al., J. Biol Chem, 271: 20399–20405 (1996); Taylor et al., J Biol Chem, 272: 10639–10645 (1997). Interferon-induced GBP's are GTPases which hydrolyze GTP to GDP and at least one GBP, and can bind to agarose-immobilized guanine nucleotides. Cheng et al., J.Biol. Chem., 258: 7746–7750 (1983). GBPs bind to GMP and GTP with similar affinity. Cheng et al., Mol Cell Biol, 11: 4717–4725 (1991); Schwemmle et al., J Biol Chem, 271: 10304–10308 (1996); Schwemmle and Staeheli, Curr Opin Cell Biol, 6: 253–259 (1994). cDNAs for chicken, rat, mouse and human GBPs have been isolated. Asundi et al., Biochim Biophys Acta, 1217:257–265 (1994); Cheng et al., (1991), supra; Schwemmle et al., J Biol Chem, 271: 10304–10308 (1996); Wynn et al., J Immunol, 147: 4384–4392 (1991). Purification of a GBP is described in Cheng et al., J. Biol. Chem., 260: 15834–15839 (1985). It was later found that two distinct interferon-alpha and -gamma-inducible genes code for two human GBPs, designated hGBP1 and hGBP2. Cheng et al., (1991), supra. Subsequently, human GBP3 was identified and partially characterized. Strehlow et al., Gene, 144: 295–299 (1994). GBP-3 was shown to have a structure related to GBP-1 and a high degree of sequence homology to both GBP-1 and GBP-2.
Schwemmle and Staeheli, J. Biol. Chem., 269: 11299–11305 (1994) showed that hGBP1, the human 67-kDa guanylate-binding protein, is a GTPase that converts GTP to GMP. Since GTP analogs with a cleavage-resistant bond between the beta- and gamma-phosphates could not be hydrolyzed by hGBP1, and pyrophosphate was no reaction product, hGBP1 seemed to degrade GTP by two consecutive cleavages of single phosphate groups. They further showed that it can be isoprenylated in vitro.
Interferon-induced GBPs have been isolated from other species such as rat (Asundi et al., Biochim. Biophys. Acta, 1217: 257–265 (1994); Vestal et al., Biochem. Biophys. Res. Commun., 224: 528–534 (1996)), murine (Vestal et al., Molecular Biology of the Cell, 7 (SUPPL.): 527A (1996) presented at the Annual Meeting of the 6th International Congress on Cell Biology and the 36th American Society for Cell Biology, San Francisco, Calif., USA, Dec. 7–11, 1996; Vestal et al., Molecular Biology of the Cell, 6 (SUPPL.): 288A (1995) presented at the Thirty-fifth Annual Meeting of the American Society for Cell Biology, Washington, D.C., USA, Dec. 9–13, 1995), and chicken. Schwemmle et al., J. Biol. Chem., 271: 10304–10308 (1996). Sequence analysis revealed that human and mouse GBPs contain only the first two elements of the typical GTP-binding consensus motif and that they contain the same sequence motif at their C termini as p21ras. Cheng et al., (1991), supra. The predicted protein sequences of these interferon-induced GBPs lack one of the three sequence motifs typically found in GTP/GDP binding proteins. The GBPs also contain carboxyl terminal residues which are substrates for post-translational modification by protein-prenyl transferases. Nantais et al., J. Leukoc Biol, 60: 423–431 (1996). Members of the Ras superfamily of GTPases are prenylated and the prenyl modification of these proteins is required for their biological function and cellular localization. Zhang and Casey, Annu Rev Biochem, 65: 241–269 (1996).
GTPases serve many different cellular functions. For example, they play key roles in such basic processes as signal transduction, vesicle transport, and translation. Most GTPases contain a tripartite GTP-binding consensus motif. Dever et al., Proc. Natl. Acad. Sci. USA, 84: 1814–1818 (1987). These sequences form part of the GTP binding pocket. The typical GTPases can assume two distinct conformations, a GTP-bound (active) and a GDP-bound (inactive) conformation. Hence, they can potentially function as molecular switches. Some GTPases, like p21ras and heterotrimeric G proteins, have a sequence motif at their C-termini that functions as an isoprenylation signal and thus ensures the proper anchoring of these proteins in cell membranes.